Complement lectin pathway components MBL and MASP-1 promote haemostasis upon vessel injury in a microvascular bleeding model.

Background Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model. Methods We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry. Results Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time. Conclusion We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system

A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model

Hemostasis-on-a-chip / incorporating the endothelium in microfluidic models of bleeding

Currently, point-of-care assays for human platelet function and coagulation are used to assess bleeding risks and drug testing, but they lack intact endothelium, a critical component of the human vascular system. Within these assays, the assessment of bleeding risk is typically indicated by the lack of or reduced platelet function and coagulation without true evaluation of hemostasis. Hemostasis is defined as the cessation of bleeding. Additionally, animal models of hemostasis also, by definition, lack human endothelium, which may limit their clinical relevance. This review discusses the current state-of-the-art of hemostasis-on-a-chip, specifically, human cell-based microfluidic models that incorporate endothelial cells, which function as physiologically relevant in vitro models of bleeding. These assays recapitulate the entire process of vascular injury, bleeding, and hemostasis, and provide real-time, direct observation, thereby serving as research-enabling tools that enhance our understanding of hemostasis and also as novel drug discovery platforms

Diabetes affects endothelial cell function and alters fibrin clot formation in a microvascular flow model: A pilot study.

Diabetes is a proinflammatory and prothrombotic condition that increases the risk of vascular complications. The aim of this study was to develop a diabetic microvascular flow model that allows to study the complex interactions between endothelial cells, blood cells and plasma proteins and their effects on clot formation. Primary human cardiac microvascular endothelial cells from donors without diabetes or donors with diabetes (type 1 or type 2) were grown in a microfluidic chip, perfused with non-diabetic or diabetic whole blood, and clot formation was assessed by measuring fibrin deposition in real time by confocal microscopy. Clot formation in non-diabetic whole blood was significantly increased in the presence of endothelial cells from donors with type 2 diabetes compared with cells from donors without diabetes. There was no significant difference in clot formation between non-diabetic and diabetic whole blood. We present for the first time a diabetic microvascular flow model as a new tool to study clot formation as a result of the complex interactions between endothelial cells, blood cells and plasma proteins in a diabetes setting. We show that endothelial cells affect clot formation in whole blood, attributing an important role to the endothelium in the development of atherothrombotic complications

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbβ3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin α(IIb)β(3) activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation